FURTHER OBSERVATIONS ON THE KOBER COLOUR AND ITTRICH FLUORESCENCE REACTIONS IN THE MEASUREMENT OF OESTRIOL, OESTRONE AND OESTRADIOL

Abstract

SUMMARY: Higher temperatures allow lower sulphuric acid concentrations and shorter heating times to be used in the Kober colour reaction for oestrogens. A one-stage reaction which is completed in 5 min. at 120° is described for oestrone and oestradiol, and a two-stage reaction which requires two periods of heating for 5 min. at 120° is described for oestriol. The conditions were applied to the Ittrich fluorescence procedure. A spectrophotofluorimetric correction was developed in which fluorescence was measured at wavelengths for excitation and emitted light near the optima for the oestrogens and at another combination at which the oestrogens produced virtually no fluorescence whereas that of impurities was not diminished. Extraction, centrifugation and fluorimetry were performed in specially designed cells. The sensitivity is 0·05–0·1 ng./sample with a linear response up to 300 ng. and a precision better than 4% in the range 1·0–100 ng.